FIGURE 3.

Assessment of inhibitors in RNA preparations. A test mastermix was prepared using the Brilliant 1-step qRT-PCR mastermix (Stratagene), to which a plant gene sense strand amplicon, primers (200 nM), and FAM-BHQ-labeled TaqMan probe (500 nM) (Applied Biosystems, Warrington, Cheshire, UK) were added. Total RNA was prepared from fresh colonic biopsies (5 mg) using Qiagen RNeasy columns and resuspended in 80 μL of Tris-Cl/EDTA buffer. RNA (100 ng) was added to the test mastermix and a one-tube RTPCR assay (final volume 25 μL, 10 hr RT at 50°C, 40 cycles of 30 min at 70°C) was carried out on a Stratagene MX-4000 real-time PCR instrument (white bars). Seven control amplifications containing water rather than RNA (black bars) were set up and run at the same time. Most of the RNA samples recorded the same C_t values as the water controls, and all but three were within 1 C_t of the control. However, one sample did record a significantly higher C_t, suggesting that this preparation contained a contaminant. Upon dilution, this sample recorded the same C_t as the control samples (not shown). C_t, threshold cycle.